The composition of human milk serves as a valuable reference for improving infant formula. Much effort has been directed at producing a milk based infant formula which is similar to human milk.
One component of human milk that is receiving more investigation is the fat composition. Human milk fat contains long chain polyunsaturated fatty acids which may play a role in infant development. Many infant formulas do not contain lipids having long chain polyunsaturated fatty acids such as arachidonic acid (C20:4w6) (referred to herein as AA), eicosapentaenoic acid (referred to herein as EPA), and docosahexaenoic acid (C22:6w3) (referred to herein as DHA). Acceptable ingredient sources for these fatty acids are limited, thus the infant formula and adult nutritional industry is in need of additional supplies of these polyunsaturated fatty acids.
Polyunsaturated acids, in particular the longer chain acids such as AA, DHA, and EPA are natural constituents of many foodstuffs in the form of glycerides (mono-, di- and tri-) or phospholipids. However these acids are either intimately combined with undesirable components such as cholesterol, phosphorus compounds, or are unsuitable for food applications in their natural form.
The n-6 family of polyunsaturated fatty acids, based on the parent linoleic acid and higher derivatives such as AA, have long been established as essential in human and animal nutrition. More recently, evidence has accumulated for the nutritional importance of the n-3 family of polyunsaturated fatty acids, based on the parent linolenic acid and higher derivatives such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These polyunsaturated acids are the precursors for prostaglandins and eicosanoids, a powerful group of compounds which produce diverse physiological actions at low concentrations. The prostaglandins are known to influence blood clotting, inflammatory and anti-inflammatory response, cholesterol absorption, bronchial function, hypertension, visual acuity and brain development in infants, and gastric secretions, among other effects.
Polyunsaturated fatty acids are found in numerous plant and animal lipid fractions. Egg yolk contains AA (arachidonic acid) and DHA (docosahexaenoic acid). Lipids isolated from egg yolks are unacceptable for use in infant formula due to high levels of cholesterol and troublesome levels of phosphorus. The AA and DHA are present in egg yolk lipids primarily as phospholipids. Thus, infant formulas fortified with egg yolk lipids have levels of cholesterol and phospholipids which far exceed the level of such compounds found in breast milk.
Typically, the amount of lipids in egg yolk is about 65% by weight (wt %) of the dry matter. In such lipids, about 66 wt % of the lipid is triglycerides, of which about 30 wt % is phospholipids, and about 4 wt % is cholesterol. The phosphorus content of the lipids is about 1 wt % to 2 wt %.
Several commercial egg lipid ingredients are presently available. The first, OVOTHIN 120, is a total egg yolk lipid extract supplied by Lucas Meyer of 765 East Pythian Ave., Decatur, Ill. 62526. OVOTHIN 120 contains triglyceride, phospholipid and cholesterol. A second ingredient, supplied by Pfanstiehl Laboratories, Inc. of 1219 Glen Rock Ave., Waukegan, Ill. 60085 is an egg yolk extract which is 90% phospholipids. Also, purified egg phospholipid is available from Genzyme Corporation of One Kendill Square, Cambridge, Mass. 02139. Unfortunately, all the above ingredients negatively impact the phosphorus levels of infant formula when used at the proper fortification level to achieve AA and DHA target levels approximating the content of AA and DHA in human milk. The proper fortification would require that about 7-9 wt % of the fat in the infant formula be composed of phospholipid. Human milk fat contains 1-3 wt % phospholipid. Furthermore, the use of a material like OVOTHIN 120 increases cholesterol in infant formula above the levels found in human milk.
There are numerous methods in the literature for recovering phospholipids from lipid mixtures. For example, U.S. Pat. No. 4,698,185 discloses a method of separating phospholipids from crude vegetable triglyceride mixtures. The method involves the addition of water in a mass ratio about equal to the mass of phospholipids present in the lipid mixture, with or without heating, and with or without co-addition of citric or phosphoric acid, to cause the phospholipids to hydrate and separate into a second phase.
Such degumming methods, however, were designed for the removal of 1 to 2 weight percent of phospholipids from crude vegetable triglycerides and are not directly applicable to the purification of other natural lipid mixtures, such as egg yolk lipids because of the higher levels of phospholipids (30-40 wt %) in eggs. Addition of a 1:1 mass ratio of water to phospholipid with large amounts of phospholipids present causes the formation of a stable emulsion which prevents phase separation. Moreover, sterols tend to partition between both the phospholipid and triglyceride phases.
It is desirable to provide a process by which cholesterol and other sterol compounds (many of which can be metabolized to cholesterol or its derivatives) can be extracted from various foodstuffs, thereby producing low-cholesterol versions of such foodstuffs. However, the process must not introduce into the foodstuff any material which is not generally recognized as safe for use in foodstuffs. In addition, the process should remove from the foodstuff not only cholesterol itself but also cholesterol derivatives and other sterol compounds which can be metabolized in the body to cholesterol or derivatives thereof, and which thus affect cholesterol levels in the body. Furthermore, the process should leave the foodstuff in a form which is as close as possible to that of the original, high cholesterol foodstuff.
Numerous attempts have previously been made to provide a cholesterol-removal process which meets these exacting criteria. U.S. Pat. No. 4,692,280, discloses a process for the purification of fish oils in which the oil is extracted with supercritical carbon dioxide to remove cholesterol, together with odoriferous and volatile impurities. Such carbon dioxide extraction processes, however, suffer from the disadvantage that they must be operated under pressure to keep the carbon dioxide in the supercritical phase, which increases the cost of the apparatus required.
U.S. Pat. No. 5,091,117 discloses a process for removing at least one sterol compound and at least one saturated fatty acid from a fluid mixture by contacting the fluid mixture with an activated charcoal. U.S. Pat. No. 5,091,117 states however, in column 12, lines 4-19, that the process should not be used for removing cholesterol from materials, such as egg yolks which contain a combination of cholesterol and proteins, since a significant adsorption of proteins and their constituent amino acids occurs on the charcoal.
British Pat. No. 1,559,064 discloses a process for removing cholesterol from butter triglycerides by distillation. However, Lanzani et al [J. Am. Oil Chem. Soc. 71, (1994) 609] determined that only 90% of the cholesterol could be removed using the process disclosed in British Pat. No. 1,559,064 without seriously affecting the quality of the end product. Excessive time at the high temperatures needed for more complete cholesterol removal was found to cause cis-trans isomerization of the polyunsaturated fatty acids. The trans form of polyunsaturated fatty acids are considered undesirable in food products.
U.S. Pat. No. 4,670,285 to M. Clandinin of Jun. 2, 1987 discloses the use of lipid extracted from egg yolk in infant formula. The lipids of the Clandinin reference include polyunsaturated lipids found in human milk such as C:20 or C:22 w6 and C:20 or C22 w3 fatty acids. The lipids of Clandinin contain the unacceptable levels of cholesterol and phosphorus of the original egg yolk material.
Abstract of JP 62198351 of Sep. 2, 1987 to Morinaga Milk discloses a substitute mothers' milk composition which contains egg yolk lipid extracted from egg yolk with ethanol. The lipid is preferably combined so that a 100 g milk composition contains 68 mg of cholesterol. However, the 68 mg of cholesterol translates to about 680 mg/L (liter) or greater than four times that found in human milk.
U.S. Pat. No. 5,112,956 of May 12, 1992 to P. Tang, et al. discloses a method for the removal of lipids and cholesterol from protein material such as that in egg yolk by treating the protein with an extraction mixture comprising a lower alcohol, water, and an acid in concentrations selected to extract cholesterol and lipids from the protein. The preferred lower alcohol of this reference is ethanol and a primary object is obtaining protein suitable for human consumption.
PTC publication WO 89/11521 of Nov. 30, 1989 discloses a process for preparing EPA and DHA and their esters from oils of animal and/or vegetable origin by subjecting the raw oil to alkaline hydrolysis, acidifying the soap so formed with a mineral acid in aqueous solution, extracting the resulting mixture with petroleum ether and after washing and concentration, the combined extracts are submitted to one or more distillation steps with the pressure and temperature parameters being suitably changed in order to obtain a whole range of desired products.
Abstract of JP 1160989 (application) of Jun. 23, 1989 to NIOF. Fresh fish eggs are extracted with solvent of distilled water, methanol/chloroform, acetone, ether, under oxygen-free conditions to extract lipids and eventually isolate a docosahexaenoic acid-containing phosphatidylcholine.
Abstract of Han'guk Ch'uksan Hakhoechi, 1991, 33(8), 602-6 by Han, C. K., et al. Egg yolk was ground with trichloromethane and methanol. Lipid extract was converted to methyl esters by transesterification with boron trifluoride and methanol. The methyl esters were analyzed for various fatty acids. C20-22 polyunsaturated acids accounted for 4.3% of the total.
In an embodiment of the present invention, egg yolk derived glyceride compositions, also simply referred to herein as Processed Natural Ingredients, are prepared which typically contain about 4 wt % of AA and about 1.5 wt % of DHA based on the weight of the Processed Natural Ingredients and wherein the amount of phosphorus can be reduced to less than about 0.002 wt % (20 ppm) and the amount of cholesterol reduced to less than about 0.1 wt % of the Processed Natural Ingredients. Preferably at least 95% and particularly at least 98% of the cholesterol and other sterols, and phosphorus compounds are removed from the lipid mixture staring material, e.g. egg yolks in the process of this invention, and such highly purified fatty acids or esters thereof are referred to herein as being "essentially free of cholesterol, sterols and phosphorus compounds". The Processed Natural Ingredients can be in the form of mono-, di-, or triglycerides as well as mixtures thereof.
Unless the context indicates otherwise, the following terms shall have the following meaning:
"AA" is arachidonic acid (C20:4w6); PA1 "alkaline metal" is an alkaline earth metal or alkali metal such as calcium, magnesium, sodium, or potassium; PA1 "DHA" is docosahexaenoic acid (C22:6w3); PA1 "egg derived triglycerides" are one of the Processed Natural Ingredients (as defined below) wherein a major portion, preferably at least 75% by weight of the glycerides and particularly at least 90% of the glycerides are triglycerides derived from egg yolk; PA1 "essentially free of cholesterol, sterols, and phosphorus compounds" means that at least 95%, preferably at least 98%, of the cholesterol and other sterols, and phosphorus compounds are removed from a lipid starting material by the process of the present invention; PA1 "FAP" is fatty acid profile; PA1 "free fatty acid route" is the process which comprises the isolation of free fatty acids by hydrolysis of naturally occurring lipid mixtures, separation of an aqueous phase from the fatty acid phase, reacting the fatty acids with the sterols to form sterol fatty acid esters and distilling the sterol fatty acid esters/fatty acid mixture; PA1 "GC" is gas chromatography; PA1 "lower alkane" is an alkane having from 1 to 4 carbon atoms; PA1 "lower alkyl" is an alkyl having from 1 to 4 carbon atoms; PA1 "lower alkanol" is a monohydric alcohol having from 1 to 4 carbon atoms; PA1 "lower alkoxide" is an alkyl oxide group having from 1 to 4 carbon atoms such as in sodium methoxide; PA1 "mL" means milliliter; PA1 "N/AP" means not applicable; PA1 "N/D" means not detectable; PA1 "N/R" means not reported; and PA1 "Processed Natural Ingredients" are the compositions containing glycerides prepared by reacting glycerol or polyhydric alcohols with the free fatty acids in the process of this invention; PA1 "TLC" is thin layer chromatography.